摘要
目的:用重组PCR技术对人单核细胞趋化蛋白鄄1(hμMCP鄄1)基因cDNA进行缺失突变,构建其突变体—7NDcDNA。方法:根据hμMCP鄄1基因缺失突变前后的两段基因分别设计两对含有酶切位点的引物,以pBluescript鄄hμMCP鄄1为模板,进行重组PCR反应,将PCR产物与T载体连接进行TA克隆,酶切鉴定并测序确定其长度为342bp,继之将目的基因克隆入pcDNA3.1真核表达载体。结果:成功构建hμMCP鄄1cDNA突变体—7ND的真核细胞表达载体,测序结果表明,该突变体N端第2至8位氨基酸缺失。结论:重组PCR技术是十分有效、可靠的基因缺失突变方法。
Objective: To construct 7ND CDNA-the deletion mutant of human monocyte chemoattractant protein-1 (MCP-1) cDNA by recombinant PCR. Methods: Using pBluescript-hμ MCP-1 as templates, two sequences before and after the mututant site were amplified with two pairs of synthetic primers. Recombinant PCR introduced the deletion mutant by linking the two sequences, and the production 7ND cDNA was cloned into T vector for further cloning. Then 7ND cDNA was inserted into plasmid pcDNA3.1 to construct 7ND eukarytic expressing vector. Results: A recombinant plasmid pcDNA3.1-7ND expressing human MCP-1 cDNA mutant was successfully constructed. The analysis of sequence proved that 7ND has a length of 342 bps and the N-terminal amino acids of 2 through 8 of human MCP-1 lacked. Conclusion: The result indicate that recombinant PCR is a very effect and reliable method for gene mutant.
出处
《山东大学学报（医学版）》
CAS
2004年第6期698-701,共4页
Journal of Shandong University:Health Sciences
基金
国家自然科学基金资助项目(60271015)
卫生部临床学科重点项目(20012943)。
关键词
单核细胞化学吸引蛋白质1
基因缺失
克隆
分子
重组聚合酶链反应
Monocyte chemoattractant protein-1
Gene deletion
Cloning, molecular
Polymerase chain reaction, recombiant
